Nitric oxide (NO) plays a major role in inflammatory diseases of the myocardium. NO is produced during viral myocarditis, transplant rejection, and post-ischemic inflammation in the heart. The major source of NO in the heart is normally endothelial nitric oxide synthase (eNOS or NOS3), which is present in endothelial cells, cardiac myocytes, and platelets. However, cytokines produced during ischemia or inflammation of the heart can induce expression of the inducible NOS (iNOS or NOS2) in a variety of cells, including cardiac myocytes and macrophages. Since iNOS produces large amounts of NO continuously, in contrast to eNOS producing smaller amounts of NO transiently, the expression of iNOS changes the concentration and location of NO in the heart. Alterations in NO production can have profound implications for cardiac physiology, since NO has a variety of effects upon the heart, including relaxation of coronary arterial smooth muscle, inhibition of cardiac myocyte contractility, reduction of platelet and leukocyte adhesion to the coronary artery wall, inhibition of glycolysis and oxidative phosphorylation, and regulation of apoptotic pathways. Because NO derived from iNOS can affect a variety of cardiac systems, the regulation of iNOS is of critical importance. Although the primary mechanism by which NOS2 is regulated was previously thought to be mediated by transcription, other mechanisms regulate NOS2 expression as well. Our preliminary data show that novel proteins regulate the stability of iNOS mRNA, and regulate the activity of iNOS protein. We propose to study transcriptional, post-transcriptional, and post- translational mechanisms of NOS2 regulation. We then will examine the physiological relevance of NOS2 regulation in a post- ischemic model of myocardial infarction. These studies will characterize novel molecular mechanisms by which radical production is regulated in ischemic myocardium.